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bioRxiv · 10.1101/2025.03.13.643086

Granzyme-targeting quenched activity-based probes for assessing tumor response to immunotherapy

Kazim, M.·Ganguly, A.·Malespini, S. M.·Thang, L.·Patel, N. L.·Kim, C.·Kalein, J. D.·Difilippantonio, S.·Yoo, E.
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Abstract

Molecular imaging of immune activation holds tremendous potential for the development of novel immunotherapy. In particular, chemical probes capable of detecting immune responses before changes in tumor size occur can guide early therapeutic strategies. Here, we present quenched activity-based probes targeting granzymes as a biomarker of antitumor immunity. Through optimization of peptide recognition element and functional chemical warhead, we have developed an optical imaging probe Cy5-IEPCyaPhP-QSY21, which rapidly reacts with GzmB at substoichiometric concentrations and enables efficient, selective labeling of the active enzyme in a complex proteome. With high specificity and minimal background signal, this probe produces GzmB-induced near-infrared fluorescence signals in the tumors of living mice shortly after injection. Both in vivo and ex vivo fluorescence signals correlate with GzmB expression and activity, and the population of CD8+ cells in tumor tissues. Moreover, it demonstrates the potential to track tumor response to immunotherapy. Thus, this study offers a chemical tool for assessing immune-mediated anticancer activity using noninvasive optical imaging. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/643086v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1bff748org.highwire.dtl.DTLVardef@3c80dorg.highwire.dtl.DTLVardef@4301ceorg.highwire.dtl.DTLVardef@12b7c8a_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Kazim, M., Ganguly, A., Malespini, S. M., Thang, L., Patel, N. L., Kim, C., Kalein, J. D., Difilippantonio, S., Yoo, E.. 2025-03-15. Granzyme-targeting quenched activity-based probes for assessing tumor response to immunotherapy. https://doi.org/10.1101/2025.03.13.643086

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